Src-homology regions 2 and 3 (SH2 and SH3) are conserved sequence motifs consisting of approximately 100 and 60 amino acid residues, respectively, and found in many eukaryotic proteins with diverse functions (1-3). SH3 domains have been identified in several cytoskeleton-associated proteins such as p80/p85, myosin 1b, and spectrin, in the neutrophil NADPH oxidase-associated proteins p47 and p67, and in several yeast proteins important for morphogenesis (Bem1p and ABP-1), mating (FUS1) or for the regulation of ras activity (Cdc25 and Ste6; for review see Musacchio et al. (4)). The observation that many SH3-containing proteins are cytoskeleton-associated led to the suggestion that SH3 domains play a role in multimeric protein complex formation at or near cytoplasmic membranes. Some proteins which contain both SH2 and SH3 domains (e.g. Grb2--the mammalian homologue of Sem5 and drk-proteins from C. elegans and drosophila, respectively) perform the function of adaptor molecules by joining activated receptor tyrosine kinases with the p21 ras guanine nucleotide-releasing protein (GNRP) SOS. Grb2 and its homologues bind to phosphotyrosine on activated membrane-anchored receptor tyrosine kinases through their SH2 domain and to SOS through their amino- and carboxyterminal SH3 domains (5-9). These processes lead to translocation of SOS to the plasma membrane where ras proteins are located. Thus, SH3-containing and SH3-binding proteins are involved in a highly conserved signal transduction pathway from activated growth factor receptors to p21 ras.
The non receptor tyrosine kinase c-src consists of an SH3, SH2 and tyrosine kinase domain. c-src appears to be most important in the normal function of osteoclasts, as determined from studies of src-knock-out mice (10). The catalytic activity of c-src and other nonreceptor tyrosine kinases is inhibited by the intramolecular association of their intrinsic SH2 domain to a phosphorylated Tyr (position 527) in the carboxy-terminal tail. Recent data indicate that the intrinsic SH3 domain (in cooperation with the SH2 domain) may also participate in the regulation of the kinase activity of these enzymes. Deletion of the c-src SH3 domain reduces the phosphorylation of Tyr-527 by csk kinase, resulting in the upregulation of c-src kinase activity (11). In addition to the above, the c-src SH3 domain may contribute to the repression of src catalytic activity by stabilizing the conformation most favorable for the interaction between the src SH2 domain and the phosphorylated carboxy-terminal Tyr-527 residue (12). Several mutations in the src SH3 domain were reported to increase its catalytic activity and oncogenicity, and there are some indications that the N-terminal region of the src SH3 domain may be responsible for specific interactions with as yet unidentified negative regulators of src activity (13). Therefore, the SH3 domain in the src family kinases can also be considered as an internal, potential regulator of kinase activity functioning in cooperation with the SH2 domain.
Several src SH3 binding proteins were isolated by affinity purification from cytoskeleton-rich fractions of Balb/c 3T3 cells, and one of these proteins was identified as paxillin, a vinculin-binding cytoskeletal protein. Some other SH3 binding proteins identified by this method possessed kinase activity, and probably belong to the family of serine and/or threonine kinases (14).
Though the structural basis for the interaction of different SH2 domains with phosphotyrosine has been well studied (15-18), the interaction between SH3 domains and SH3 binding proteins is much less well characterized, partly because only a few SH3 binding proteins have been identified. The first reported SH3 binding protein (3BP-1) bound to the SH3 domain of the c-abl tyrosine kinase (and to the SH3 domain of c-src as well) through a proline-rich sequence (19). This protein was identified by using a glutathione S-transferase (GST) fusion protein, which included the SH3 region of the c-Abl proto oncogene, to probe a .lambda.gt11 cDNA expression library. The SH3 binding sequences in 3BP-1 and several other proteins identified in this work were localized to a nine or ten amino acid proline-rich motif, XPXXPPP.PSI.XP (SEQ ID NO: 1, .PSI. represents hydrophobic amino acid residues) (20). Similar motifs have since been recognized in several other proteins including the PI 3-kinase p85 subunit (21), dynamin (22), formin, and the acetylcholine muscarinic receptor (20). In several of these proteins the putative binding sites are multiple and overlapping (22).
The specificity of SH3 binding proteins toward different SH3 domains has been studied using the 3BP-1 protein (with Abl, Src, Neural Src, Crk-SH3 domains, (19)), dynamin (with 15 different SH3 domains, (22)), paxillin (with Src, neural Src, Lyn SH3-domains, (14)). These experiments demonstrate that most of the presently identified SH3 domain binding sequences have a broad spectrum of SH3 domains as possible binding partners (19, 22). On the other hand, no binding motifs have been identified for some SH3 domains which exhibit a low homology to the Src SH3 domain (e.g. the ras-GAP SH3 domain). Therefore, the identification of SH3 binding clones and identification of motifs recognized by SH3 domains represents an important step in understanding the determinants of specificity for this type of protein-protein interaction.
Random peptide libraries offer a unique, abundant and complex source of short peptides which can be used to identify specific binding sequences and core amino acid consensus sequences for virtually any screening agent. Successful screening of these libraries has been described not only for epitopes recognized by monoclonal antibodies (23-33), but also for the identification of peptide sequences interacting directly with other proteins such as the molecular chaperone BiP (34), calmodulin (35), a.sub.5 b.sub.1 integrin (36), platelet glycoprotein IIb/IIIa (37), S-Protein (15, 38) streptavidin (39) and concanavalin A (40, 41).
Recently, Chen et al. (42) utilized the SH3 domain of PI3-kinase p85 to screen a biased combinatorial library of synthetic peptides in which prolines were fixed in three of nine positions with the six other positions being randomized. The bias for this library, represented by the formula XXXPPXPXX (SEQ ID NO: 2), was derived from an alignment of the SH3-binding motifs in 3BP-1 and the guanine nucleotide exchange factor Sos1.
Various strategies have been employed to screen phage cDNA libraries for clones encoding proteins which interact with the screening agent. For example, phage cDNA libraries have been screened with antibodies, nucleic acids, and tyrosine-phosphorylated polypeptides. The identification of cDNA clones encoding proteins which interact with src SH3 domain, native (intact) c-src, or any segment of c-src has never previously been reported.